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Compositions for in vitro amplification of nucleic acids

Inactive Publication Date: 2005-06-09
QUANTA BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012] The instant invention relates to the use of anti-foam agents in enzymatic reactions, in particular in in vitro nucleic acid amplification reactions and, more particularly, in homogenous phase or real time reactions that exploit optical detection of a fluorescent signal to quantify and detect amplification product. The present invention provides methods and compositions for improving the accuracy of optical detection in real time PCR by eliminating interfering factors such as bubbles commonly encountered in real time PCR. The invention also provides methods of detecting the amplification of a target nucleic acid that reduces variation in background fluorescence. The invention also provides kits for enzymatic reactions, including kits for amplification of nucleic acid sequence, that incorporate the anti-foam moiety described herein.

Problems solved by technology

Both these approaches are time consuming and require multiple amplification reactions to quantify a specific nucleic acid sequence present in a single sample.
A major problem in automating PCR data analysis is identification of baseline fluorescence.
These problems are often exacerbated by bubbles in the reaction that interfere with optical measurements.
A major problem in understanding of gene expression patterns for gene discovery and identification of metabolic pathways is the limitations of current methods for accurate quantification.
Use of real time PCR methods provides a significant improvement towards this goal, however, limitations in accurate liquid handling and delivery for assembly of real time PCR reactions still present a significant problem.
This problem is exacerbated, moreover, when dealing with small volumes required for high through put analyses.

Method used

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  • Compositions for in vitro amplification of nucleic acids
  • Compositions for in vitro amplification of nucleic acids
  • Compositions for in vitro amplification of nucleic acids

Examples

Experimental program
Comparison scheme
Effect test

example i

Real-Time TaqMan PCR in the Presence of Varying Amounts of Anti-Foam

[0077] This example demonstrates the ability of PCR to proceed in the presence of an anti-foam compound.

[0078] Real-time quantitative polymerase chain reactions specific for the human cytoplasmic β-actin sequence were carried out using a commercial hot-start Taq DNA polymerase reaction cocktail as follows. Each 50 μl PCR contained 1×iQ PCR SuperMix (Bio-Rad Laboratories), 200 nM each primer, and 100 nM FAM and TAMRA labeled 5′-nuclease probe as described by Xu et al. 2000, Focus 22:3-5 (forward primer: 5′-CCTGGCACCCAGCACAAT-3′; reverse primer: 5′-GGGCCGGACTCGTCATAC-3′; Taqman probe: 5′-FAM-AGCCGCCGATCCACACGGAGT-TAMRA-3′), and varying amounts of a DNA target. Triplicate PCRs were performed for each amount of input DNA (1×102, 1×104, 1×106, or 1×108 copies of a plasmid containing the gene encoding human cytoplasmic β-actin). PCRs were conducted under identical conditions except for the inclusion of varying amounts (...

example ii

Real-Time TaqMan PCR in the Presence of Different Anti-Foam Compounds

[0081] This example demonstrates the ability of PCR to proceed in the presence of a variety of anti-foam compounds.

[0082] Real-time quantitative polymerase chain reactions specific for the human cytoplasmic β-actin sequence were carried out as described above in example 1. PCRs were conducted under identical conditions except for the inclusion of 0.005% of anti-foam selected from different commercially available preparations from Dow Corning (1520-US, AF, or FG-10) or Sigma (O-30, SE-15, or Antifoam B). Control reactions omitted anti-foam. Results are summarized in table 2.

[0083] Results demonstrated that a variety of anti-foams with different chemical compositions, fatty acid ester (Sigma O-30), or silicone emulsions comprised of polydimethylsiloxane, and emulsion formulations are effective at suppressing foaming by PCR surfactants without adverse effect on DNA amplification. For all anti-foams tested, the pres...

example iii

Real-Time SYBR Green I PCR in the Presence of Antifoam Reagent

[0084] This example extends the efficacy and compatibility of anti-foam in real-time PCR using different homogeneous detection chemistries, specifically, fluorescent dsDNA-specific dyes.

[0085] Real-time quantitative polymerase chain reactions specific for the human cytoplasmic β-actin sequence were carried out using a commercial hot-start Taq DNA polymerase reaction cocktail modified for use with SYBR Green I as follows. Each 50 μl PCR contained 1×iQ PCR SuperMix (Bio-Rad Laboratories), 2% DMSO, 0.25×SYBR Green I (Molecular Probes), 300 nM each primer, as described by Xu et al. 2000, Focus 22:3-5 (forward primer: 5′-CCTGGCACCCAGCACAAT-3′; reverse primer: 5′-GGGCCGGACTCGTCATAC-3′), and varying amounts of a DNA target. Triplicate PCRs were performed for each amount of input DNA (1×102, 1×104, 1×106, or 1×108 copies of a plasmid containing the gene encoding human cytoplasmic β-actin). PCRs were conducted under identical co...

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Abstract

Method and compositions for improving DNA polymerase and reverse transcriptase reactions are provided. Addition of anti-foam reagents to the reactions improves fluid handling, especially of small volumes and allows enhanced accuracy of optical detection, without substantially inhibiting enzymatic activity.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 60 / 400,685, filed Aug. 5, 2002, the contents of which are hereby incorporate by reference in their entirety.FIELD OF THE INVENTION [0002] The invention provides improved methods for detecting and amplifying nucleic acid molecules. More specifically, the invention provides methods for nucleic acid amplification that employ anti-foam reagents to improve fluidic handling and provide enhanced accuracy of real-time optical monitoring of amplification reaction mixtures. BACKGROUND [0003] The polymerase chain reaction (PCR) is a fundamental technique in molecular biology for the amplification of nucleic acid sequences in biological samples (Mullis, K. et al., Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich H. et al., EP 50,424; EP 84,796, EP 258,017, EP 237,362; Mullis, K., EP 201,184; Mullis K. et al., U.S. Pat. No. 4,683,202; Erlich, H., U.S. Pat. No. 4,582,788; and Saiki, R. et al., U.S. Pat. ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2527/125
Inventor RASHTCHIAN, AYOUBSCHUSTER, DAVID
Owner QUANTA BIOSCI
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